Formulations for coated microprojections containing non-volatile counterions

ABSTRACT

The invention provides for a formulation for coating one or more microprojections which reduces or minimizes the loss of counterions from the coating in order to achieve a pH-stabilized formulation.

This application claims the benefit of U.S. Provisional Application No.60/484,020, filed Jun. 30, 2003.

FIELD OF THE PRESENT INVENTION

This invention relates to administering and enhancing the transdermaldelivery of an agent across the skin. More particularly, the inventionrelates to a percutaneous drug delivery system for administering abiologically active agent through the stratum corneum using skinpiercing microprojections which have a dry coating of the biologicallyactive agent. Delivery of the agent is achieved when themicroprojections pierce the skin of a patient and the patient'sinterstitial fluid contacts and dissolves the active agent. Morespecifically it relates to a coating formulation which resists changesin the pH of the coating and promotes the solubilization of the coatingafter the microprojections have pierced the skin.

BACKGROUND OF THE INVENTION

Drugs are most conventionally administered either orally or byinjection. Unfortunately, many medicaments are completely ineffective orhave radically reduced efficacy when orally administered since theyeither are not absorbed or are adversely affected before entering thebloodstream and thus do not possess the desired activity. On the otherhand, the direct injection of the medicament into the bloodstream, whileassuring no modification of the medicament during administration, is adifficult, inconvenient, painful and uncomfortable procedure, sometimesresulting in poor patient compliance.

Hence, in principle, transdermal delivery provides for a method ofadministering drugs that would otherwise need to be delivered viahypodermic injection or intravenous infusion. Transdermal drug deliveryoffers improvements in both of these areas. Transdermal delivery whencompared to oral delivery avoids the harsh environment of the digestivetract, bypasses gastrointestinal drug metabolism, reduces first-passeffects, and avoids the possible deactivation by digestive and liverenzymes. Conversely, the digestive tract is not subjected to the drugduring transdermal administration. Indeed, many drugs such as aspirinhave an adverse effect on the digestive tract. However, in manyinstances, the rate of delivery or flux of many agents via the passivetransdermal route is too limited to be therapeutically effective.

The word “transdermal” is used herein as a generic term referring topassage of an agent across the skin layers. The word “transdermal”refers to delivery of an agent (e.g., a therapeutic agent such as adrug) through the skin to the local tissue or systemic circulatorysystem without substantial cutting or piercing of the skin, such ascutting with a surgical knife or piercing the skin with a hypodermicneedle. Transdermal agent delivery includes delivery via passivediffusion as well as by external energy sources including electricity(e.g., iontophoresis) and ultrasound (e.g., phonophoresis). While drugsdo diffuse across both the stratum corneum and the epidermis, the rateof diffusion through the stratum corneum is often the limiting step.Many compounds, in order to achieve a therapeutic dose, require higherdelivery rates than can be achieved by simple passive transdermaldiffusion. When compared to injections, transdermal agent deliveryeliminates the associated pain and reduces the possibility of infection.

Theoretically, the transdermal route of agent administration could beadvantageous in the delivery of many therapeutic proteins, becauseproteins are susceptible to gastrointestinal degradation and exhibitpoor gastrointestinal uptake and transdermal devices are more acceptableto patients than injections. However, the transdermal flux of medicallyuseful peptides and proteins is often insufficient to be therapeuticallyeffective due to the large size/molecular weight of these molecules.Often the delivery rate or flux is insufficient to produce the desiredeffect or the agent is degraded prior to reaching the target site, forexample while in the patient's bloodstream.

Transdermal drug delivery systems generally rely on passive diffusion toadminister the drug while active transdermal drug delivery systems relyon an external energy source (e.g., electricity) to deliver the drug.Passive transdermal drug delivery systems are more common. Passivetransdermal systems have a drug reservoir containing a highconcentration of drug adapted to contact the skin where the drugdiffuses through the skin and into the body tissues or bloodstream of apatient. The transdermal drug flux is dependent upon the condition ofthe skin, the size and physical/chemical properties of the drugmolecule, and the concentration gradient across the skin. Because of thelow permeability of the skin to many drugs, transdermal delivery has hadlimited applications. This low permeability is attributed primarily tothe stratum corneum, the outermost skin layer which consists of flat,dead cells filled with keratin fibers (keratinocytes) surrounded bylipid bilayers. This highly-ordered structure of the lipid bilayersconfers a relatively impermeable character to the stratum corneum.

Active transport systems use an external energy source to assist drugflux through the stratum corneum. One such enhancement for transdermaldrug delivery is referred to as “electrotransport.” This mechanism usesan electrical potential, which results in the application of electriccurrent to aid in the transport of the agent through a body surface,such as skin. Other active transport systems use ultrasound(phonophoresis) and heat as the external energy source.

There also have been many attempts to mechanically penetrate or disruptthe outermost skin layers thereby creating pathways into the skin inorder to enhance the amount of agent being transdermally delivered.Early vaccination devices known as scarifiers generally had a pluralityof tines or needles which are applied to the skin to scratch or makesmall cuts in the area of application. The vaccine was applied eithertopically on the skin, such as U.S. Pat. No. 5,487,726 issued to Rabenauor as a wetted liquid applied to the scarifier tines such as U.S. Pat.No. 4,453,926, issued to Galy or U.S. Pat. No. 4,109,655 issued toChacornac, or U.S. Pat. No. 3,136,314 issued to Kravitz. Scarifiers havebeen suggested for intradermal vaccine delivery in part because onlyvery small amounts of the vaccine need to be delivered into the skin tobe effective in immunizing the patient. Further, the amount of vaccinedelivered is not particularly critical since an excess amount achievessatisfactory immunization as well as a minimum amount. However a seriousdisadvantage in using a scarifier to deliver a drug is the difficulty indetermining the transdermal drug flux and the resulting dosagedelivered. Also due to the elastic, deforming and resilient nature ofskin to deflect and resist puncturing, the tiny piercing elements oftendo not uniformly penetrate the skin and/or are wiped free of a liquidcoating of an agent upon skin penetration. Additionally, due to the selfhealing process of the skin, the punctures or slits made in the skintended to close up after removal of the piercing elements from thestratum corneum. Thus, the elastic nature of the skin acts to remove theactive agent coating which has been applied to the tiny piercingelements upon penetration of these elements into the skin. Furthermorethe tiny slits formed by the piercing elements heal quickly afterremoval of the device, thus limiting the passage of agent through thepassageways created by the piercing elements and in turn limiting thetransdermal flux of such devices.

Other devices which use tiny skin piercing elements to enhancetransdermal drug delivery are disclosed in European Patent EP 0407063A1,U.S. Pat. No. 5,879,326 issued to Godshall, et al., U.S. Pat. No.3,814,097 issued to Ganderton, et al., U.S. Pat. No. 5,279,544 issued toGross, et al., U.S. Pat. No. 5,250,023 issued to Lee, et al., U.S. Pat.No. 3,964,482 issued to Gerstel, et al., Reissue 25,637 issued toKravitz, et al., and PCT Publication Nos. WO 96/37155, WO 96/37256, WO96/17648, WO 97/03718, WO 98/11937, WO 98/00193, WO 97/48440, WO97/48441, WO 97/48442, WO 98/00193, WO 99/64580, WO 98/28037, WO98/29298, and WO 98/29365; all incorporated by reference in theirentirety. These devices use piercing elements of various shapes andsizes to pierce the outermost layer (i.e., the stratum corneum) of theskin. The piercing elements disclosed in these references generallyextend perpendicularly from a thin, flat member, such as a pad or sheet.The piercing elements in some of these devices are extremely small, somehaving dimensions (i.e., a microblade length and width) of only about25-400 μm and a microblade thickness of only about 5-50 μm. These tinypiercing/cutting elements make correspondingly smallmicroslits/microcuts in the stratum corneum for enhanced transdermalagent delivery therethrough.

Generally, these systems include a reservoir for holding the drug andalso a delivery system to transfer the drug from the reservoir throughthe stratum corneum, such as by hollow tines of the device itself.Another alternative is to provide a coating containing the active agenton the microprojections themselves. Such an approach has been disclosedin published U.S. patent application Ser. Nos. 2002/0132054,2002/0193729, 2002/0177839, and 2002/0128599, all which are incorporatedherein by reference.

Using a microprojection device to transdermally deliver an agent coatedon the microprojections confers a number of benefits. However, some ofthe formulations used for coating the microprojections do not achieve acoating that is readily solubilized upon piercing the skin.

Accordingly, it is an object of the invention to provide a coating thathas improved solubility.

It is another object of the invention to provide a coating thatstabilizes the pH of the coating and can increase the amount ofuncharged biologically active agent, which is less soluble inphysiological fluids.

SUMMARY OF THE INVENTION

In accordance with the above objects and those that will be mentionedand will become apparent below, the device and method for transdermallydelivering a biologically active agent in accordance with this inventiongenerally comprises a delivery system having a microprojection member(or system) that includes at least one microprojection (or arraythereof) that are adapted to pierce through the stratum corneum into theunderlying epidermis layer, or epidermis and dermis layers. In oneembodiment, the microprojection includes a biocompatible coating havingat least one biologically active agent disposed therein.

As such, one embodiment of the invention is a composition for coating atransdermal delivery device having stratum corneum-piercingmicroprojections comprising a formulation of a biologically active agentand a non-volatile counterion, wherein said formulation has increased pHstability and solubility when dried.

Suitable biologically active agents include therapeutic agents in allthe major therapeutic areas including, but not limited to:anti-infectives, such as antibiotics and antiviral agents; analgesics,including fentanyl, sufentanil, remifentanil, buprenorphine andanalgesic combinations; anesthetics; anorexics; antiarthritics;antiasthmatic agents, such as terbutaline; anticonvulsants;antidepressants; antidiabetic agents; antidiarrheals; antihistamines;anti-inflammatory agents; antimigraine preparations; antimotion sicknesspreparations such as scopolamine and ondansetron; antinauseants;antineoplastics; antiparkinsonism drugs; antipruritics; antipsychotics;antipyretics; antispasmodics, including gastrointestinal and urinary;anticholinergics; sympathomimetrics; xanthine derivatives;cardiovascular preparations, including calcium channel blockers such asnifedipine; beta blockers; beta-agonists such as dobutamine andritodrine; antiarrythmics; antihypertensives, such as atenolol; ACEinhibitors, such as ranitidine; diuretics; vasodilators, includinggeneral, coronary, peripheral, and cerebral; central nervous systemstimulants; cough and cold preparations; decongestants; diagnostics;hormones, such as parathyroid hormone; hypnotics; immunosuppressants;muscle relaxants; parasympatholytics; parasympathomimetrics;prostaglandins; proteins; peptides; psychostimulants; sedatives; andtranquilizers. Other suitable agents include vasoconstrictors,anti-healing agents and pathway patency modulators.

Further specific examples of agents include, without limitation, growthhormone release hormone (GHRH), growth hormone release factor (GHRF),insulin, insultropin, calcitonin, octreotide, endorphin, thyroliberin(TRH), NT-36 (chemical name:N-[[(s)-4-oxo-2-azetidinyl]carbonyl]-L-histidyl-L-prolinamide),liprecin, pituitary hormones (e.g., human growth hormone (HGH),menotropin (hMG), desmopressin acetate, etc), follicle luteoids, alphanatriuretic factor (aANF), growth factors such as growth factorreleasing factor (GFRF), beta-melanocyte-stimulating hormone (bMSH),growth hormone (GH), somatostatin, bradykinin, somatotropin,platelet-derived growth factor releasing factor, asparaginase, bleomycinsulfate, chymopapain, cholecystokinin, chorionic gonadotropin,erythropoietin, epoprostenol (platelet aggregation inhibitor), glucagon,human chorionic gonadotropin (HCG), hirulog, hyaluronidase, interferonalpha, interferon beta, interferon gamma, interleukins, interleukin-10(IL-10), erythropoietin (EPO), granulocyte macrophage colony stimulatingfactor (GM-CSF), granulocyte colony stimulating factor (G-CSF),glucagon, leutinizing hormone releasing hormone (LHRH), LHRH analogs(such as goserelin, leuprolide, buserelin, triptorelin, gonadorelin, andnapfarelin, menotropins (urofollitropin (follicle stimulating hormone(FSH) and leutinizing hormone (LH))), oxytocin, streptokinase, tissueplasminogen activator, urokinase, vasopressin, deamino [Val4, D-Arg8]arginine vasopressin, desmopressin, corticotropin (adrenocorticotropichormone (ACTH)), ACTH analogs such as ACTH (1-24), atrial natriureticpeptide (ANP), ANP clearance inhibitors, angiotensin II antagonists,antidiuretic hormone agonists, bradykinin antagonists, ceredase,corticostatin analogs (CSI's), calcitonin gene related peptide (CGRP),enkephalins, FAB fragments, IgE peptide suppressors, insulin-like growthfactor-1 (IGF-1), neurotrophic factors, colony stimulating factors,parathyroid hormone and agonists, parathyroid hormone antagonists,parathyroid hormone (PTH), PTH analogs such as PTH (1-34), prostaglandinantagonists, pentigetide, protein C, protein S, renin inhibitors,thymosin alpha-1, thrombolytics, tumor necrosis factor (TNF),vasopressin antagonists analogs, alpha-1 antitrypsin (recombinant), andtransforming growth factor-beta (TGF-beta).

The biologically active agent can also comprise a vaccine, includingviruses and bacteria, protein-based vaccines, polysaccharide-basedvaccine, nucleic acid-based vaccines, and other antigenic agents.Suitable antigenic agents include, without limitation, antigens in theform of proteins, polysaccharide conjugates, oligosaccharides, andlipoproteins. These subunit vaccines in include Bordetella pertussis(recombinant PT accince-acellular), Clostridium tetani (purified,recombinant), Corynebacterium diptheriae (purified, recombinant),Cytomegalovirus (glycoprotein subunit), Group A streptococcus(glycoprotein subunit, glycoconjugate Group A polysaccharide withtetanus toxoid, M protein/peptides linke to toxing subunit carriers, Mprotein, multivalent type-specific epitopes, cysteine protease, C5apeptidase), Hepatitis B virus (recombinant Pre S 1, Pre-S2, S,recombinant core protein), Hepatitis C virus (recombinant-expressedsurface proteins and epitopes), Human papillomavirus (Capsid protein,TA-GN recombinant protein L2 and E7 [from HPV-6], MEDI-501 recombinantVLP L1 from HPV-11, Quadrivalent recombinant BLP L1 [from HPV-6],HPV-11, HPV-16, and HPV-18, LAMP-E7 [from HPV-16]), Legionellapneumophila (purified bacterial survace protein), Neisseria meningitides(glycoconjugate with tetanus toxoid), Pseudomonas aeruginosa (syntheticpeptides), Rubella virus (synthetic peptide), Streptococcus pneumoniae(glyconconjugate [1, 4, 5, 6B, 9N, 14, 18C, 19V, 23F] conjugated tomeningococcal B OMP, glycoconjugate [4, 6B, 9V, 14, 18C, 19F, 23F]conjugated to CRM197, glycoconjugate [1, 4, 5, 6B, 9V, 14, 18C, 19F,23F] conjugated to CRM1970, Treponema pallidum (surface lipoproteins),Varicella zoster virus (subunit, glycoproteins), and Vibrio cholerae(conjugate lipopolysaccharide).

Whole virus or bacteria include, without limitation, weakened or killedviruses, such as cytomegalo virus, hepatitis B virus, hepatitis C virus,human papillomavirus, rubella virus, and varicella zoster, weakened orkilled bacteria, such as bordetella pertussis, clostridium tetani,corynebacterium diptheriae, group A streptococcus, legionellapneumophila, neisseria meningitdis, pseudomonas aeruginosa,streptococcus pneumoniae, treponema pallidum, and vibrio cholerae, andmixtures thereof.

Additional commercially available vaccines, which contain antigenicagents, include, without limitation, flu vaccines, lyme disease vaccine,rabies vaccine, measles vaccine, mumps vaccine, chicken pox vaccine,small pox vaccine, hepatitus vaccine, pertussis vaccine, and diptheriavaccine.

Vaccines comprising nucleic acids include, without limitation,single-stranded and double-stranded nucleic acids, such as, for example,supercoiled plasmid DNA; linear plasmid DNA; cosmids; bacterialartificial chromosomes (BACs); yeast artificial chromosomes (YACs);mammalian artificial chromosomes; and RNA molecules, such as, forexample, mRNA. The size of the nucleic acid can be up to thousands ofkilobases. In addition, in certain embodiments of the invention, thenucleic acid can be coupled with a proteinaceous agent or can includeone or more chemical modifications, such as, for example,phosphorothioate moieties. The encoding sequence of the nucleic acidcomprises the sequence of the antigen against which the immune responseis desired. In addition, in the case of DNA, promoter andpolyadenylation sequences are also incorporated in the vaccineconstruct. The antigen that can be encoded include all antigeniccomponents of infectious diseases, pathogens, as well as cancerantigens. The nucleic acids thus find application, for example, in thefields of infectious diseases, cancers, allergies, autoimmune, andinflammatory diseases.

Suitable immune response augmenting adjuvants which, together with thevaccine antigen, can comprise the vaccine include aluminum phosphategel; aluminum hydroxide; algal glucan: b-glucan; cholera toxin Bsubunit; CRL1005: ABA block polymer with mean values of x=8 and y=205;gamma inulin: linear (unbranched) β-D(2->1)polyfructofuranoxyl-a-D-glucose; Gerbu adjuvant: N-acetylglucosamine-(b1-4)-N-acetylmuramyl-L-alanyl-D-glutamine (GMDP), dimethyldioctadecylammonium chloride (DDA), zinc L-proline salt complex(Zn-Pro-8); Imiquimod(1-(2-methypropyl)-1H-imidazo[4,5-c]quinolin-4-amine; ImmTherÔ:N-acetylglucoaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glyceroldipalmitate; MTP-PE liposomes: C59H108N6O19PNa-3H20 (MTP); Murametide:Nac-Mur-L-Ala-D-Gln-OCH3; Pleuran: b-glucan; QS-21; S-28463: 4-amino-a,a-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanol; sclavo peptide:VQGEESNDK.HCl (IL-1b 163-171 peptide); and threonyl-MDP (TermurtideÔ):N-acetyl muramyl-L-threonyl-D-isoglutamine, and interleukine 18, IL-2IL-12, IL-15, Adjuvants also include DNA oligonucleotides, such as, forexample, CpG containing oligonucleotides. In addition, nucleic acidsequences encoding for immuno-regulatory lymphokines such as IL-18, IL-2IL-12, IL-15, IL-4, IL10, gamma interferon, and NF kappa B regulatorysignaling proteins can be used.

Generally, in the noted embodiments of the invention, the amount ofcounterion should neutralize the charge of the biologically activeagent. In such embodiments, the counterion or the mixture of counterionis present in amounts necessary to neutralize the charge present on theagent at the pH of the formulation. Excess of counterion (as the freeacid or as a salt) can be added to the peptide in order to control pHand to provide adequate buffering capacity.

In another preferred embodiment the counterion is a strong acid. Strongacids can be defined as presenting at least one pKa lower than about 2.Examples of such acids include hydrochloric acid, hydrobromic acid,nitric acid, sulfonic acid, sulfuric acid, maleic acid, phosphoric acid,benzene sulfonic acid and methane sulfonic acid.

Another preferred embodiment is directed to a mixture of counterionswherein at least one of the counterion is a strong acid and at least oneof the counterion is a low volatility weak acid.

Another preferred embodiment is directed to a mixture of counterionswherein at least one of the counterion is a strong acid and at least oneof the counterion is a weak acid with high volatility. Volatile weakacid counterions present at least one pKa higher than about 2 and amelting point lower than about 50° C. or a boiling point lower thanabout 170° C. at P_(atm). Examples of such acids include acetic acid,propionic acid, pentanoic acid and the like.

The acidic counterion is present in amounts necessary to neutralize thepositive charge present on the drug at the pH of the formulation. Excessof counterion (as the free acid or as a salt) can be added to the drugin order to control pH and to provide adequate buffering capacity.

In one embodiment of the invention, the coating formulations include atleast one antioxidant, which can be sequestering agents such sodiumcitrate, citric acid, EDTA (ethylene-dinitrilo-tetraacetic acid) or freeradical scavengers such as ascorbic acid, methionine, sodium ascorbate,and the like.

In one embodiment of the invention, the coating formulation includes atleast one surfactant, which can be zwitterionic, amphoteric, cationic,anionic, or nonionic, including, without limitation, sodiumlauroamphoacetate, sodium dodecyl sulfate (SDS), cetylpyridiniumchloride (CPC), dodecyltrimethyl ammonium chloride (TMAC), benzalkonium,chloride, polysorbates such as TWEEN® 20 (polyoxyethylene-20-sorbitanmonolaurate) and TWEEN® 80 (polyoxyethylene-80-sorbitan monolaurate)TWEEN 80, other sorbitan derivatives, such as sorbitan laurate, andalkoxylated alcohols, such as laureth-4.

In a further embodiment of the invention, the coating formulationincludes at least one polymeric material or polymer that has amphiphilicproperties, which can comprise, without limitation, cellulosederivatives, such as hydroxyethylcellulose (HEC),hydroxypropylmethylcell-ulose (HPMC), hydroxypropycellulose (HPC),methylcellulose (MC), hydroxyethylmethylcellulose (HEMC), orethylhydroxy-ethylcellulose (EHEC), as well as PLURONIC® typecopolymers.

In another embodiment, the coating formulation includes a hydrophilicpolymer selected from the following group: hydroxyethyl starch, dextran,poly(vinyl alcohol), poly(ethylene oxide),poly(2-hydroxyethylmethacrylate), poly(n-vinyl pyrolidone), polyethyleneglycol and mixtures thereof, and like polymers.

In another embodiment of the invention, the coating formulation includesa biocompatible carrier, which can comprise, without limitation, humanalbumin, bioengineered human albumin, polyglutamic acid, polyasparticacid, polyhistidine, pentosan polysulfate, polyamino acids, sucrose,trehalose, melezitose, raffinose and stachyose.

In another embodiment, the coating formulation includes a stabilizingagent, which can comprise, without limitation, a non-reducing sugar, apolysaccharide or a reducing sugar. Suitable non-reducing sugars for usein the methods and compositions of the invention include, for example,sucrose, trehalose, stachyose, or raffinose. Suitable polysaccharidesfor use in the methods and compositions of the invention include, forexample, dextran, soluble starch, dextrin, and insulin. Suitablereducing sugars for use in the methods and compositions of the inventioninclude, for example, monosaccharides such as, for example, apiose,arabinose, lyxose, ribose, xylose, digitoxose, fucose, quercitol,quinovose, rhamnose, allose, altrose, fructose, galactose, glucose,gulose, hamamelose, idose, mannose, tagatose, and the like; anddisaccharides such as, for example, primeverose, vicianose, rutinose,scillabiose, cellobiose, gentiobiose, lactose, lactulose, maltose,melibiose, sophorose, and turanose, and the like.

In another embodiment, the coating formulation includes avasoconstrictor, which can comprise, without limitation, amidephrine,cafaminol, cyclopentamine, deoxyepinephrine, epinephrine, felypressin,indanazoline, metizoline, midodrine, naphazoline, nordefrin, octodrine,omipressin, oxymethazoline, phenylephrine, phenylethanolamine,phenylpropanolamine, propylhexedrine, pseudoephedrine, tetrahydrozoline,tramazoline, tuaminoheptane, tymazoline, vasopressin, xylometazoline andthe mixtures thereof. The most preferred vasoconstrictors includeepinephrine, naphazoline, tetrahydrozoline indanazoline, metizoline,tramazoline, tymazoline, oxymetazoline and xylometazoline.

In another embodiment of the invention, the coating formulation includesat least one “pathway patency modulator”, which can comprise, withoutlimitation, osmotic agents (e.g., sodium chloride), zwitterioniccompounds (e.g., amino acids), and anti-inflammatory agents, such asbetamethasone 21-phosphate disodium salt, triamcinolone acetonide21-disodium phosphate, hydrocortamate hydrochloride, hydrocortisone21-phosphate disodium salt, methylprednisolone 21-phosphate disodiumsalt, methylprednisolone 21-succinaate sodium salt, paramethasonedisodium phosphate and prednisolone 21-succinate sodium salt, andanticoagulants, such as citric acid, citrate salts (e.g., sodiumcitrate), dextrin sulfate sodium, aspirin and EDTA.

In yet another embodiment of the invention, the coating formulationincludes a solubilizing/complexing agent, which can compriseAlpha-Cyclodextrin, Beta-Cyclodextrin, Gamma-Cyclodextrin,glucosyl-alpha-Cyclodextrin, maltosyl-alpha-Cyclodextrin,glucosyl-beta-Cyclodextrin, maltosyl-beta-Cyclodextrin, hydroxypropylbeta-cyclodextrin, 2-hydroxypropyl-beta-Cyclodextrin,2-hydroxypropyl-gamma-Cyclodextrin, hydroxyethyl-beta-Cyclodextrin,methyl-beta-Cyclodextrin, sulfobutylether-alpha-cyclodextrin,sulfobutylether-beta-cyclodextrin, andsulfobutylether-gamma-cyclodextrin. Most preferredsolubilizing/complexing agents are beta-cyclodextrin, hydroxypropylbeta-cyclodextrin, 2-hydroxypropyl-beta-Cyclodextrin andsulfobutylether7 beta-cyclodextrin.

In another embodiment of the invention, the coating formulation includesat least one non-aqueous solvent, such as ethanol, isopropanol,methanol, propanol, butanol, propylene glycol, dimethysulfoxide,glycerin, N,N-dimethylformamide and polyethylene glycol 400.

Preferably, the coating formulations have a viscosity less thanapproximately 500 centipoise and greater than 3 centipoise.

In one embodiment of the invention, the thickness of the biocompatiblecoating is less than 25 microns, more preferably, less than 10 microns.

The invention also comprises transdermal delivery devices having atleast one microprojection configured to pierce the stratum corneumcoated with the noted formulations.

In one embodiment of the invention, the device has a microprojectiondensity of at least approximately 10 microprojections/cm², morepreferably, in the range of at least approximately 200-2000microprojections/cm².

In one embodiment, the microprojection is constructed out of stainlesssteel, titanium, nickel titanium alloys, or similar biocompatiblematerials.

In another embodiment, the microprojection is constructed out of anon-conductive material, such as a polymer. Alternatively, themicroprojection can be coated with a non-conductive material, such asParylene®, or a hydrophobic material, such as Teflon®, silicon or otherlow energy material.

Generally, the methods of the invention comprise applying a coating of abiologically active agent to a transdermal delivery device, wherein thetransdermal delivery device comprises a plurality of stratumcorneum-piercing microprojections, comprising the steps of providing aformulation of the biologically active agent, stabilizing theformulation by adding a non-volatile counterion, and applying saidformulation to the microprojections. Preferably, the counterion is addedin an amount to neutralize the charge on the biologically active agent.The charge of the agent can be determined using the algorithms of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in greater detail with reference tothe preferred embodiments illustrated in the accompanying drawings andfigures wherein:

FIG. 1 is a graph showing the charge profile of acetic acid (pKa 4.75)as a function of pH;

FIG. 2 is a graph showing the mole ratios of uncharged acetic acid andcharged acetate ion as a function of pH;

FIG. 3 is a graph showing the charge profile of fentanyl as a functionof pH.

FIG. 4 is a graph showing the mole ratios of the neutral (Fentanyl base)and charged (Fentanyl+1) fentanyl species as a function of pH;

FIG. 5 is a graph showing the charge profile of hPTH(1-34) as a functionof pH;

FIG. 6 is a graph showing the mole ratios of the net charged species ofhPTH(1-34) as a function of pH;

FIG. 7 is a graph showing the mole ratios of fentanyl acetate, aceticacid and the neutral form of fentanyl (Fentanyl base) as a function ofpH;

FIG. 8 is a graph showing the mole ratios for acetic acid the netneutral form of hPTH(1-34) as function of pH;

FIG. 9 is a graph showing the charge profile of a peptide comprising ahGRF analog;

FIG. 10 is a diagram showing the depicting the loss of volatilecounterion from the outer layer of a coating;

FIG. 11 is a perspective view of a microprojection array that would beused in conjunction with the present invention; and

FIG. 12 is a perspective view of a microprojection array showing severalmicroprojections that have been coated.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless stated otherwise the following terms used herein have thefollowing meanings.

The term “transdermal” means the delivery of an agent into and/orthrough the skin for local or systemic therapy.

The term “transdermal flux” means the rate of transdermal delivery.

The term “co-delivering” as used herein, means that a supplementalagent(s) is administered transdermally either before the agent isdelivered, before and during transdermal flux of the agent, duringtransdermal flux of the agent, during and after transdermal flux of theagent, and/or after transdermal flux of the agent. Additionally, two ormore agents may be coated onto the microprojections resulting inco-delivery of the agents.

The term “biologically active agent” or “active agent” as used herein,refers to a composition of matter or mixture containing a drug which ispharmacologically effective when administered in a therapeuticallyeffective amount.

Such agents include therapeutic agents in all the major therapeuticareas including, but not limited to: anti-infectives such as antibioticsand antiviral agents; analgesics, including fentanyl, sufentanil,remifentanil, buprenorphine and analgesic combinations; anesthetics;anorexics; antiarthritics; antiasthmatic agents such as terbutaline;anticonvulsants; antidepressants; antidiabetic agents; antidiarrheals;antihistamines; anti-inflammatory agents; antimigraine preparations;antimotion sickness preparations such as scopolamine and ondansetron;antinauseants; antineoplastics; antiparkinsonism drugs; antipruritics;antipsychotics; antipyretics; antispasmodics, including gastrointestinaland urinary; anticholinergics; sympathomimetrics; xanthine derivatives;cardiovascular preparations, including calcium channel blockers such asnifedipine; beta blockers; beta-agonists such as dobutamine andritodrine; antiarrythmics; antihypertensives such as atenolol; ACEinhibitors such as ranitidine; diuretics; vasodilators, includinggeneral, coronary, peripheral, and cerebral; central nervous systemstimulants; cough and cold preparations; decongestants; diagnostics;hormones such as parathyroid hormone; hypnotics; immunosuppressants;muscle relaxants; parasympatholytics; parasympathomimetrics;prostaglandins; proteins; peptides; psychostimulants; sedatives; andtranquilizers. Other suitable agents include vasoconstrictors,anti-healing agents and pathway patency modulators.

Further specific examples of agents include, without limitation, growthhormone release hormone (GHRH), growth hormone release factor (GHRF),insulin, insultropin, calcitonin, octreotide, endorphin, thyroliberin(TRH), NT-36 (chemical name:N-[[(s)-4-oxo-2-azetidinyl]carbonyl]-L-histidyl-L-prolinamide),liprecin, pituitary hormones (e.g., human growth hormone (HGH),menotropin (hMG), desmopressin acetate, etc), follicle luteoids, alphanatriuretic factor (aANF), growth factors such as growth factorreleasing factor (GFRF), beta-melanocyte-stimulating hormone (bMSH),growth hormone (GH), somatostatin, bradykinin, somatotropin,platelet-derived growth factor releasing factor, asparaginase, bleomycinsulfate, chymopapain, cholecystokinin, chorionic gonadotropin,erythropoietin, epoprostenol (platelet aggregation inhibitor), glucagon,human chorionic gonadotropin (HCG), hirulog, hyaluronidase, interferonalpha, interferon beta, interferon gamma, interleukins, interleukin-10(IL-10), erythropoietin (EPO), granulocyte macrophage colony stimulatingfactor (GM-CSF), granulocyte colony stimulating factor (G-CSF),glucagon, leutinizing hormone releasing hormone (LHRH), LHRH analogs(such as goserelin, leuprolide, buserelin, triptorelin, gonadorelin, andnapfarelin, menotropins (urofollitropin (follicle stimulating hormone(FSH) and leutinizing hormone (LH))), oxytocin, streptokinase, tissueplasminogen activator, urokinase, vasopressin, deamino [Val4, D-Arg8]arginine vasopressin, desmopressin, corticotropin (adrenocorticotropichormone (ACTH)), ACTH analogs such as ACTH (1-24), atrial natriureticpeptide (ANP), ANP clearance inhibitors, angiotensin II antagonists,antidiuretic hormone agonists, bradykinin antagonists, ceredase,corticostatin analogs (CSI's), calcitonin gene related peptide (CGRP),enkephalins, FAB fragments, IgE peptide suppressors, insulin-like growthfactor-1 (IGF-1), neurotrophic factors, colony stimulating factors,parathyroid hormone and agonists, parathyroid hormone antagonists,parathyroid hormone (PTH), PTH analogs such as PTH (1-34), prostaglandinantagonists, pentigetide, protein C, protein S, renin inhibitors,thymosin alpha-1, thrombolytics, tumor necrosis factor (TNF),vasopressin antagonists analogs, alpha-1 antitrypsin (recombinant), andtransforming growth factor-beta (TGF-beta).

The term “biologically active agent” or “active agent” as used hereinalso refers to a composition of matter or mixture containing a vaccineor other immunologically active agent or an agent which is capable oftriggering the production of an immunologically active agent, and whichis directly or indirectly immunologically effective when administered ina immunologically effective amount.

Suitable vaccines include viruses and bacteria, protein-based vaccines,polysaccharide-based vaccine, nucleic acid-based vaccines, and otherantigenic agents. Suitable antigenic agents include, without limitation,antigens in the form of proteins, polysaccharide conjugates,oligosaccharides, and lipoproteins. Subunit vaccines include Bordetellapertussis (recombinant pertussis toxin vaccine—acellular), Clostridiumtetani (purified, recombinant), Corynebacterium diptheriae (purified,recombinant), Cytomegalovirus (glycoprotein subunit), Group Astreptococcus (glycoprotein subunit, glycoconjugate Group Apolysaccharide with tetanus toxoid, M protein/peptides linked to toxinsubunit carriers, M protein, multivalent type-specific epitopes,cysteine protease, C5a peptidase), Hepatitis B virus (recombinant PreS1, Pre-S2, S, recombinant core protein), Hepatitis C virus(recombinant-expressed surface proteins and epitopes), Humanpapillomavirus (Capsid protein, TA-GN recombinant protein L2 and E7[from HPV-6], MEDI-501 recombinant VLP L1 from HPV-11, Quadrivalentrecombinant BLP L1 [from HPV-6], HPV-11, HPV-16, and HPV-18, LAMP-E7[from HPV-16]), Legionella pneumophila (purified bacterial surfaceprotein), Neisseria meningitides (glycoconjugate with tetanus toxoid),Pseudomonas aeruginosa (synthetic peptides), Rubella virus (syntheticpeptide), Streptococcus pneumoniae (glyconconjugate [1, 4, 5, 6B, 9N,14, 18C, 19V, 23F] conjugated to meningococcal B OMP, glycoconjugate [4,6B, 9V, 14, 18C, 19F, 23F] conjugated to CRM197, glycoconjugate [1, 4,5, 6B, 9V, 14, 18C, 19F, 23F] conjugated to CRM1970, Treponema pallidum(surface lipoproteins), Varicella zoster virus (subunit, glycoproteins),and Vibrio cholerae (conjugate lipopolysaccharide).

Whole virus or bacteria include, without limitation, weakened or killedviruses, such as cytomegalo virus, hepatitis B virus, hepatitis C virus,human papillomavirus, rubella virus, and varicella zoster, weakened orkilled bacteria, such as bordetella pertussis, clostridium tetani,corynebacterium diptheriae, group A streptococcus, legionellapneumophila, neisseria meningitdis, pseudomonas aeruginosa,streptococcus pneumoniae, treponema pallidum, and vibrio cholerae, andmixtures thereof.

Additional commercially available vaccines, which contain antigenicagents, include, without limitation, flu vaccines, lyme disease vaccine,rabies vaccine, measles vaccine, mumps vaccine, chicken pox vaccine,small pox vaccine, hepatitus vaccine, pertussis vaccine, and diptheriavaccine.

Vaccines comprising nucleic acids include, without limitation,single-stranded and double-stranded nucleic acids, such as, for example,supercoiled plasmid DNA; linear plasmid DNA; cosmids; bacterialartificial chromosomes (BACs); yeast artificial chromosomes (YACs);mammalian artificial chromosomes; and RNA molecules, such as, forexample, mRNA. The size of the nucleic acid can be up to thousands ofkilobases. In addition, in certain embodiments of the invention, thenucleic acid can be coupled with a proteinaceous agent or can includeone or more chemical modifications, such as, for example,phosphorothioate moieties. The encoding sequence of the nucleic acidcomprises the sequence of the antigen against which the immune responseis desired. In addition, in the case of DNA, promoter andpolyadenylation sequences are also incorporated in the vaccineconstruct. The antigen that can be encoded include all antigeniccomponents of infectious diseases, pathogens, as well as cancerantigens. The nucleic acids thus find application, for example, in thefields of infectious diseases, cancers, allergies, autoimmune, andinflammatory diseases.

Suitable immune response augmenting adjuvants which, together with thevaccine antigen, can comprise the vaccine include aluminum phosphategel; aluminum hydroxide; algal glucan: b-glucan; cholera toxin Bsubunit; CRL1005: ABA block polymer with mean values of x=8 and y=205;gamma inulin: linear (unbranched) β-D(2->1)polyfructofuranoxyl-a-D-glucose; Gerbu adjuvant: N-acetylglucosamine-(b1-4)-N-acetylmuramyl-L-alanyl-D-glutamine (GMDP), dimethyldioctadecylammonium chloride (DDA), zinc L-proline salt complex(Zn-Pro-8); Imiquimod(1-(2-methypropyl)-1H-imidazo[4,5-c]quinolin-4-amine; ImmTherÔ:N-acetylglucoaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glyceroldipalmitate; MTP-PE liposomes: C59H108N6O19PNa-3H20 (MTP); Murametide:Nac-Mur-L-Ala-D-Gln-OCH3; Pleuran: b-glucan; QS-21; S-28463: 4-amino-a,a-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanol; sclavo peptide:VQGEESNDK.HCl (IL-1b 163-171 peptide); and threonyl-MDP (TermurtideÔ):N-acetyl muramyl-L-threonyl-D-isoglutamine, and interleukine 18, IL-2IL-12, IL-15, Adjuvants also include DNA oligonucleotides, such as, forexample, CpG containing oligonucleotides. In addition, nucleic acidsequences encoding for immuno-regulatory lymphokines such as IL-18, IL-2IL-12, IL-15, IL-4, IL10, gamma interferon, and NF kappa B regulatorysignaling proteins can be used.

It is to be understood that more than one agent may be incorporated intothe agent formulation in the method of this invention, and that the useof the term “active agent” in no way excludes the use of two or moresuch agents or drugs. The agents can be in various forms, such as freebases, acids, charged or uncharged molecules, components of molecularcomplexes or nonirritating, pharmacologically acceptable salts. Also,simple derivatives of the agents (such as ethers, esters, amides, etc)which are easily hydrolyzed at body pH, enzymes, etc., can be employed.

The term “biologically effective amount” or “biologically effectiverate” shall be used when the biologically active agent is apharmaceutically active agent and refers to the amount or rate of thepharmacologically active agent needed to effect the desired therapeutic,often beneficial, result. The amount of agent employed in the coatingswill be that amount necessary to deliver a therapeutically effectiveamount of the agent to achieve the desired therapeutic result. Inpractice, this will vary widely depending upon the particularpharmacologically active agent being delivered, the site of delivery,the severity of the condition being treated, the desired therapeuticeffect and the dissolution and release kinetics for delivery of theagent from the coating into skin tissues. It is not practical to definea precise range for the therapeutically effective amount of thepharmacologically active agent incorporated into the microprojectionsand delivered transdermally according to the methods described herein.

The term “biologically effective amount” or “biologically effectiverate” may also be used when the biologically active agent is animmunologically active agent and refers to the amount or rate of theimmunologically active agent needed to stimulate or initiate the desiredimmunologic, often beneficial result. The amount of the immunologicallyactive agent employed in the coatings will be that amount necessary todeliver an amount of the agent needed to achieve the desiredimmunological result. In practice, this will vary widely depending uponthe particular immunologically active agent being delivered, the site ofdelivery, and the dissolution and release kinetics for delivery of theagent from the coating into skin tissues.

The term “microprojections” refers to piercing elements which areadapted to pierce or cut through the stratum corneum into the underlyingepidermis layer, or epidermis and dermis layers, of the skin of a livinganimal, particularly a human. The piercing elements should not piercethe skin to a depth which causes bleeding. Typically the piercingelements have a blade length of less than 500 μm, and preferably lessthan 250 μm. The microprojections typically have a width of about 10 to200 μm and thickness of about 5 to 50 μm. The microprojections may beformed in different shapes, such as needles, hollow needles, blades,pins, punches, and combinations thereof.

The term “microprojection array” as used herein, refers to a pluralityof microprojections arranged in an array for piercing the stratumcorneum. The microprojection array may be formed by etching or punchinga plurality of microprojections from a thin sheet and folding or bendingthe microprojections out of the plane of the sheet to form aconfiguration such as that shown in FIG. 11. The microprojection arraymay also be formed in other known manners, such as by forming one ormore strips having microprojections along an edge of each of thestrip(s) as disclosed in Zuck, U.S. Pat. No. 6,050,988. Themicroprojection array may include hollow needles which hold a drypharmacologically active agent.

The term “polyelectrolyte” as used herein, means formulations ofbiologically active agents having ionic species. A polyelectrolyte is amacromolecular substance which, on dissolving in water or anotherionizing solvent, dissociates to give multiply charged anions orcations. For example, agents comprising polypeptides frequently havecomplex ionic characters resulting from multiple amino acid residueshaving acidic and basic functionalities.

Volatile counterions are defined as weak acids presenting at least onepKa higher than about 2 and a melting point lower than about 50° C. or aboiling point lower than about 170° C. at P_(atm). Examples of suchacids include acetic acid, propionic acid, pentanoic acid and the like.Volatile counterions are also defined as weak bases presenting at leastone pKa lower than about 12 and a melting point lower than about 50° C.or a boiling point lower than about 170° C. at P_(atm). Examples of suchbases include ammonia and morpholine.

Non-volatile counterions are defined as weak acids presenting at leastone acidic pKa and a melting point higher than about 50° C. or a boilingpoint higher than about 170° C. at P_(atm). Examples of such acidsinclude citric acid, succinic acid, glycolic acid, gluconic acid,glucuronic acid, lactic acid, malic acid, pyruvic acid, tartaric acid,tartronic acid, and fumaric acid. Non-volatile counterions are alsodefined as acidic zwitterions presenting at least two acidic pKa, and atleast one basic pKa, so that there is at least one extra acidic group ascompared to the number of basic groups. Examples of such compoundsinclude glutamic acid and aspartic acid.

Non-volatile counterions are also defined as weak bases presenting atleast one basic pKa and a melting point higher than about 50° C. or aboiling point higher than about 170° C. at P_(atm). Examples of suchbases include monoethanolomine, diethanolamine, triethanolamine,tromethamine, methylglucamine, glucosamine. Non-volatile counterions arealso defined as basic zwitterions presenting at least one acidic pKa,and at least two basic pKa's, wherein the number of basic pKa's isgreater than the number of acidic pka's. Examples of such compoundsinclude histidine, lysine, and arginine.

Non-volatile counterions are also defined as strong acids presenting atleast one pKa lower than about 2. Examples of such acids includehydrochloric acid, hydrobromic acid, nitric acid, sulfonic acid,sulfuric acid, maleic acid, phosphoric acid, benzene sulfonic acid andmethane sulfonic acid. Non-volatile counterions are further defined asstrong bases presenting at least one pKa higher than about 12. Examplesof such bases include sodium hydroxide, potassium hydroxide, calciumhydroxide, and magnesium hydroxide.

When referring to the volatility of a counterion, reference will alwaysbe made to the volatility of the non-ionized form of the counterion(e.g. acetic acid versus acetate).

Some drugs behave like strong bases or strong acids (for examplequaternary ammonium salts such as clidinium bromide or glycopyrrolate,sulfate derivatives such as pentosan polysulfate, some phosphoricderivatives such as nucleic acids) and are totally ionized in a widerange of pH (i.e., 4-10) that is commonly used to manufacturepharmaceutical formulations. Other compounds, such as neutralpolysaccharides (ie., insulin and dextrans), do not present acidic orbasic functions. For these classes of compounds, solubility in water isnot significantly affected by pH, and the invention does not apply.

Conversely, many drugs behave as weak acids or weak bases. Their neutralforms usually present low water solubility. For example the neutral formof many small molecular compounds such as fentanyl or peptides such ashPTH(1-34) are notoriously insoluble in water. These compounds exhibitmaximum solubility in water when they are in an electrically chargedstate. Because of their weakly acidic or basic nature, the respectiveconcentrations of the neutral and ionized forms and, hence, thesolubility in water, is pH dependant. The invention applies to thisclass of drugs. As will be evident from the examples discussed below,combination of this type of drug with a non-volatile counterion inratios sufficient to minimize the presence of the neutral form of thedrug assures water solubility of the drug in the formulation, stabilityduring storage in the solid state, and dissolution in the biologicalfluids at the time of administration.

References to the area of the sheet or member and reference to someproperty per area of the sheet or member are referring to the areabounded by the outer circumference or border of the sheet.

The term “pattern coating” refers to coating an agent onto selectedareas of the microprojections. More than one agent may be pattern coatedonto a single microprojection array. Pattern coatings can be applied tothe microprojections using known micro-fluid dispensing techniques suchas micropipeting and ink jet coating.

The drugs that will benefit from this invention contain at least oneweak acidic and/or one weak basic function and are present as a neutralspecies in the pH range pH 4 to pH 10. The mole ratio between theuncharged species and the charged species should be at least 1 to 100 inthis pH range.

The non-volatile counterion is present in an amount sufficient to reducethe mole ratio between the uncharged species and the charged species ofthe drug to less than about 1 to 100.

The present invention is based upon the discovery that coatings madefrom formulations that incorporate volatile counterions will lose thevolatile counterions from the outer surface of the coating. This resultsin a shift in the pH of the coating and can increase the amount ofuncharged biologically active agent, which is less soluble inphysiological fluids.

The present invention provides a coating formulation containing abiologically active agent which when coated and dried upon one or moremicroprojections forms a coating which reduces the loss of counterionsfrom the coating, stabilized the pH of the coating and enhances thesolubilization of the coating upon insertion into the skin. The presentinvention further includes a device having a plurality of stratumcorneum-piercing microprojections extending therefrom. Themicroprojections are adapted to pierce through the stratum corneum intothe underlying epidermis layer, or epidermis and dermis layers, but donot penetrate so deep as to reach the capillary beds and causesignificant bleeding. The microprojections have a dry coating thereonwhich contains the biologically active agent. The coating is formulatedto reduce, minimize and/or eliminate loss of volatile counter ions fromthe coating which enhances solubilization of the coating upon piercingthe skin. Upon piercing the stratum corneum layer of the skin, theagent-containing coating is dissolved by body fluid (intracellularfluids and extracellular fluids such as interstitial fluid) and releasedinto the skin for local or systemic therapy.

The solid coating is obtained by drying a formulation on themicroprojection, as described in U.S. patent application Publication No.2002/0128599. The formulation is usually an aqueous formulation. Duringthe drying process, all volatiles, including water are mostly removed(the final solid coating still contains up to about 10% water). If avolatile compound that is in equilibrium between its ionized andnon-ionized forms is present in solution, only the non-ionized formdisappears from the formulation at the time where the drying processtakes place and the ionized form stays in solution and incorporated intothe coating.

In a solid coating on a microprojection array, the drug is typicallypresent in an amount of less than about 1 mg per unit dose. With theaddition of excipients and counterions, the total mass of solid coatingis less than 3 mg per unit dose. The array is usually present on anadhesive backing, which is attached to a disposable polymeric retainerring. This assembly is packaged individually in a pouch or a polymerichousing.

In addition to the assembly, this package contains an atmosphere(usually inert) that represents a volume of at least 3 mL. This largevolume (as compared to that of the coating) acts as a sink for anyvolatile component. For example, at 20° C., the amount of acetic acidpresent in a 3 mL atmosphere as a result of its vapor pressure would beabout 0.15 mg. This amount is typically what would be present in thesolid coating if acetic acid were used as a counterion. In addition,components of the assembly such as the adhesive are likely to act asadditional sinks for volatile components. As a result, during long-termstorage, it is likely that the concentration of any volatile componentpresent in the coating would change dramatically.

The above conditions are atypical of traditional packaging ofpharmaceutical compounds where large amounts of excipients are usuallypresent. Even with very potent biotechnology compounds that arelyophilized for use as injectable, very large excess of buffers andexcipients are present in the dry cake. Thus the effect of loss ofvolatile counterion-ions does not effect the solubilization of thesetraditional dosage forms.

In the case of a drug of interest bearing a positive charge at thedesired pH, the counterion is an acid. In a preferred embodiment, theacidic counterion is a non-volatile weak acid. In another preferredembodiment, the counterion is a non-volatile strong acid.

Another preferred embodiment is directed to a mixture of counterionswherein at least one of the counterions is a strong acid and at leastone of the counterion is a non-volatile weak acid.

Another preferred embodiment is directed to a mixture of counterionswherein at least one of the counterions is a non-volatile acid and atleast one of the counterions is a weak acid with high volatility.

The acidic counterion is present in amounts necessary to neutralize thepositive charge present on the drug at the pH of the formulation. Excessof counterion (as the free acid or as a salt) can be added to the drugin order to control pH and to provide adequate buffering capacity.

In the case of a drug of interest bearing a negative charge at thedesired pH, the counterion is a base.

In a preferred embodiment, the basic counterion is a weak base with lowvolatility.

In another preferred, embodiment the counterion is a strong base.

Another preferred embodiment is directed to a mixture of counterionswherein at least one of the counterions is a strong base and at leastone of the counterions is a weak base with low volatility.

Another preferred embodiment is directed to a mixture of counterionswherein at least one of the counterions is a non-volatile base and atleast one of the counterions is a weak base with high volatility.

The basic counterion is present in amounts necessary to neutralize thenegative charge present on the drug of interest at the pH of theformulation. Excess of counterion (as the free base or as a salt) can beadded to the drug in order to control pH and to provide adequatebuffering capacity.

The present invention relates to a pharmaceutical dosage form which is asolid coating applied to one or more microprojections on amicroprojection array. The coating contains an ionized drug which has atleast one weak and/or one basic functional group

The kinetics of the agent-containing coating dissolution and releasewill depend on many factors including the nature of the drug, thecoating process, the coating thickness and the coating composition(e.g., the presence of coating formulation additives). Depending on therelease kinetics profile, it may be necessary to maintain the coatedmicroprojections in piercing relation with the skin for extended periodsof time (e.g., up to about 8 hours). This can be accomplished byanchoring the delivery device to the skin using adhesives or by usinganchored microprojections, such as described in WO 97/48440,incorporated by reference in its entirety.

FIG. 11 illustrates one embodiment of a stratum corneum-piercingmicroprojection transdermal delivery device for use with the presentinvention. FIG. 11 shows a portion of a device 10 having a plurality ofmicroprojections 12. The microprojections 12 extend at substantially a90° angle from a sheet 14 having openings 16. The sheet 14 may beincorporated in a delivery patch including a backing for the sheet 14and may additionally include adhesive for adhering the patch to theskin. In this embodiment, the microprojections are formed by etching orpunching a plurality of microprojections from a thin metal sheet andbending the microprojections out of a plane of the sheet. Metals such asstainless steel and titanium are preferred. Metal microprojections aredisclosed in Trautman et al, U.S. Pat. No. 6,083,196; Zuck U.S. Pat. No.6,050,988; and Daddona et al., U.S. Pat. No. 6,091,975; the disclosuresof which are incorporated herein by reference. Other microprojectionsthat can be used with the present invention are formed by etchingsilicon using silicon chip etching techniques or by molding plasticusing etched micro-molds. Silicon and plastic microprojections aredisclosed in Godshall et al., U.S. Pat. No. 5,879,326, the disclosuresof which are incorporated herein by reference.

FIG. 12 illustrates the microprojection transdermal delivery devicehaving microprojections 12 having a biologically active agent-containingcoating 18. The coating 18 may partially or completely cover themicroprojections. For example, the coating can be in a dry patterncoating on the microprojections. The coatings can be applied before orafter the microprojections are formed.

The coating on the microprojections can be formed by a variety of knownmethods. One such method is dip-coating. Dip-coating can be described asa means to coat the microprojections by partially or totally immersingthe microprojections into the drug-containing coating solution.Alternatively the entire device can be immersed into the coatingsolution. Coating only those portions the microprojection that piercesthe skin is preferred.

By use of the partial immersion technique described above, it ispossible to limit the coating to only the tips of the microprojections.There is also a roller coating mechanism that limits the coating to thetips of the microprojection. This technique is described in a U.S.patent application Ser. No. 10/099,604, filed 15 Mar. 2002, which isfully incorporated herein by reference.

Other coating methods include spraying the coating solution onto themicroprojections. Spraying can encompass formation of an aerosolsuspension of the coating composition. In a preferred embodiment anaerosol suspension forming a droplet size of about 10 to 200 picolitersis sprayed onto the microprojections and then dried. In anotherembodiment, a very small quantity of the coating solution can bedeposited onto the microprojections as a pattern coating 18. The patterncoating 18 can be applied using a dispensing system for positioning thedeposited liquid onto the microprojection surface. The quantity of thedeposited liquid is preferably in the range of 0.5 to 20nl/microprojection. Examples of suitable precision metered liquiddispensers are disclosed in U.S. Pat. Nos. 5,916,524; 5,743,960;5,741,554; and 5,738,728, the disclosures of which are incorporatedherein by reference. Microprojection coating solutions can also beapplied using ink jet technology using known solenoid valve dispensers,optional fluid motive means and positioning means which is generallycontrolled by use of an electric field. Other liquid dispensingtechnology from the printing industry or similar liquid dispensingtechnology known in the art can be used for applying the pattern coatingof this invention.

In all cases, after a coating has been applied, the coating solution isdried onto the microprojections by various means. In a preferredembodiment the coated device is dried in ambient room conditions.However, various temperatures and humidity levels can be used to dry thecoating solution onto the microprojections. Additionally, the devicescan be heated, lyophilized, freeze dried or similar techniques used toremove the water from the coating.

A number of factors affect the volatility of compounds. These includetemperature, atmospheric pressure, and vapor pressure of the compound.The volatilization process is time dependant. In addition, ionizedcompounds present a much lower volatility as compared to their unionizedforms. For example, acetic acid has a boiling point of 118° C. whilesodium acetate is essentially non-volatile. If a volatile compound inequilibrium between its ionized and non-ionized forms is present in asolution, only the non-ionized form disappears from the solution and theionized form stays in solution.

If the volatile compound is a weak acid AH the following equilibriumtakes place in solution:AH

A⁻+H⁺

With Ka1 being the equilibrium constant for AH, the equilibrium can bewritten as:Ka1=(A ⁻)×(H ⁺)/(AH)

(A⁻), (H⁺) and (AH) represent the concentrations of the species presentin solution.

If AH is volatile, the equilibrium will shift towards A⁻+H⁺

AH in order to satisfy the laws of equilibrium. Ultimately, the entiremass of the volatile weak acid will disappear from the solution.

If the volatile compound is a weak base (B) the following equilibriumtakes place:B+H⁺

BH⁺

With Ka2 being the equilibrium constant, the equilibrium can be writtenas:Ka2=(B)×(H ⁺)/(BH ⁺).

(B), (H⁺), and (BH⁺) represent the concentrations of the species presentin solution.

If B is volatile, the equilibrium will shift towards BH⁺

B+H⁺ in order to satisfy the laws of equilibrium. Ultimately, the entiremass of the volatile weak base will disappear from the solution.

When a weak acid and a weak base are mixed in solution, they form a saltaccording to the following equilibrium:AH+B

A⁻+BH⁺

With Ka1 and Ka2 representing the equilibrium constants for AH and B,respectively, the equilibrium can be written as:Ka1/Ka2=(A ⁻)×(BH ⁺)/(AH)×(B).

If AH is volatile, the equilibrium will shift towards A⁻+BH⁺

AH+B in order to satisfy the laws of equilibrium. The net result will bean increase in the concentration of the free base and a resultingincrease in pH.

Conversely, if B is volatile, the equilibrium will shift identicallywith a net result of an increase in the concentration of the free acidand a decrease in pH. Strong acids present a particular case becausemany of them are highly volatile. Indeed, hydrochloric acid is a gas inambient conditions. When combined with a base, they form non-volatilesalts because they are completely ionized in a wide pH range with theexception of extreme pH for some acids. In solution, or in the solidstate, volatilization of the counterion occurs at the interface betweenthe solution and the atmosphere or the solid and the atmosphere. In asolution, the high diffusivity of solutes minimizes differences inconcentration between the interface and the bulk of the solution.

Conversely, in a solid state, diffusivity is very slow or non-existentand greater concentration gradients of the volatile counterion areachieved between the interface and the bulk of the solution. Ultimately,the outer layer of the coating is depleted in counterion while the bulkof the solid coating is relatively unchanged as compared to the initialdry state (see FIG. 10). This situation may results in a highlyinsoluble outer coating if the counterion is associated with a drug thatis substantially insoluble in its neutral net charge state. Indeed, aswill be explained in detail in Example 1, volatilization of thecounterion results in formation of the water insoluble neutral species.This, in turn, jeopardizes dissolution of the drug from the solidcoating upon exposure to the biological fluids.

Other known formulation adjuvants can be added to the coating solutionas long as they do not adversely affect the necessary solubility andviscosity characteristics of the coating solution and the physicalintegrity of the dried coating.

Generally, in the noted embodiments of the invention, the amount ofcounterion should neutralize the charge of the biologically activeagent. In such embodiments, the counterion or the mixture of counterionis present in amounts necessary to neutralize the charge present on theagent at the pH of the formulation. Excess of counterion (as the freeacid or as a salt) can be added to the peptide in order to control pHand to provide adequate buffering capacity.

In another preferred embodiment the counterion is a strong acid. Strongacids can be defined as presenting at least one pKa lower than about 2.Examples of such acids include hydrochloric acid, hydrobromic acid,nitric acid, sulfonic acid, sulfuric acid, maleic acid, phosphoric acid,benzene sulfonic acid and methane sulfonic acid.

Another preferred embodiment is directed to a mixture of counterionswherein at least one of the counterion is a strong acid and at least oneof the counterion is a low volatility weak acid.

Another preferred embodiment is directed to a mixture of counterionswherein at least one of the counterions is a strong acid and at leastone of the counterion is a weak acid with high volatility. Volatile weakacid counterions present at least one pKa higher than about 2 and amelting point lower than about 50° C. or a boiling point lower thanabout 170° C. at P_(atm). Examples of such acids include acetic acid,propionic acid, pentanoic acid and the like.

The acidic counterion is present in amounts necessary to neutralize thepositive charge present on the drug at the pH of the formulation. Excessof counterion (as the free acid or as a salt) can be added to the drugin order to control pH and to provide adequate buffering capacity.

In one embodiment of the invention, the coating formulations include atleast one antioxidant, which can be sequestering agents such sodiumcitrate, citric acid, EDTA (ethylene-dinitrilo-tetraacetic acid) or freeradical scavengers such as ascorbic acid, methionine, sodium ascorbate,and the like. Presently preferred antioxidants include EDTA andmethionine.

In one embodiment of the invention, the coating formulation includes atleast one surfactant, which can be zwitterionic, amphoteric, cationic,anionic, or nonionic, including, without limitation, sodiumlauroamphoacetate, sodium dodecyl sulfate (SDS), cetylpyridiniumchloride (CPC), dodecyltrimethyl ammonium chloride (TMAC), benzalkonium,chloride, polysorbates such as TWEEN® 20 and TWEEN® 80, other sorbitanderivatives, such as sorbitan laurate, and alkoxylated alcohols, such aslaureth-4.

In a further embodiment of the invention, the coating formulationincludes at least one polymeric material or polymer that has amphiphilicproperties, which can comprise, without limitation, cellulosederivatives, such as hydroxyethylcellulose (HEC),hydroxypropylmethylcell-ulose (HPMC), hydroxypropycellulose (HPC),methylcellulose (MC), hydroxyethylmethylcellulose (HEMC), orethylhydroxy-ethylcellulose (EHEC), as well as PLURONIC® typecopolymers.

In another embodiment, the coating formulation includes a hydrophilicpolymer selected from the following group: hydroxyethyl starch, dextran,poly(vinyl alcohol), poly(ethylene oxide),poly(2-hydroxyethylmethacrylate), poly(n-vinyl pyrolidone), polyethyleneglycol and mixtures thereof, and like polymers.

In another embodiment of the invention, the coating formulation includesa biocompatible carrier, which can comprise, without limitation, humanalbumin, bioengineered human albumin, polyglutamic acid, polyasparticacid, polyhistidine, pentosan polysulfate, polyamino acids, sucrose,trehalose, melezitose, raffinose and stachyose.

In another embodiment, the coating formulation includes a stabilizingagent, which can comprise, without limitation, a non-reducing sugar, apolysaccharide or a reducing sugar. Suitable non-reducing sugars for usein the methods and compositions of the invention include, for example,sucrose, trehalose, stachyose, or raffinose. Suitable polysaccharidesfor use in the methods and compositions of the invention include, forexample, dextran, soluble starch, dextrin, and insulin. Suitablereducing sugars for use in the methods and compositions of the inventioninclude, for example, monosaccharides such as, for example, apiose,arabinose, lyxose, ribose, xylose, digitoxose, fucose, quercitol,quinovose, rhamnose, allose, altrose, fructose, galactose, glucose,gulose, hamamelose, idose, mannose, tagatose, and the like; anddisaccharides such as, for example, primeverose, vicianose, rutinose,scillabiose, cellobiose, gentiobiose, lactose, lactulose, maltose,melibiose, sophorose, and turanose, and the like.

In another embodiment, the coating formulation includes avasoconstrictor, which can comprise, without limitation, amidephrine,cafaminol, cyclopentamine, deoxyepinephrine, epinephrine, felypressin,indanazoline, metizoline, midodrine, naphazoline, nordefrin, octodrine,ornipressin, oxymethazoline, phenylephrine, phenylethanolamine,phenylpropanolamine, propylhexedrine, pseudoephedrine, tetrahydrozoline,tramazoline, tuaminoheptane, tymazoline, vasopressin, xylometazoline andthe mixtures thereof. The most preferred vasoconstrictors includeepinephrine, naphazoline, tetrahydrozoline indanazoline, metizoline,tramazoline, tymazoline, oxymetazoline and xylometazoline.

As will be appreciated by one having ordinary skill in the art, theaddition of a vasoconstrictor to the coating formulations and, hence,solid biocompatible coatings of the invention is particularly useful toprevent bleeding that can occur following application of themicroprojection device or array and to prolong the pharmacokinetics ofthe active agent through reduction of the blood flow at the applicationsite and reduction of the absorption rate from the skin site into thesystem circulation.

In another embodiment of the invention, the coating formulation includesat least one “pathway patency modulator”, which can comprise, withoutlimitation, osmotic agents (e.g., sodium chloride), zwitterioniccompounds (e.g., amino acids), and anti-inflammatory agents, such asbetamethasone 21-phosphate disodium salt, triamcinolone acetonide21-disodium phosphate, hydrocortamate hydrochloride, hydrocortisone21-phosphate disodium salt, methylprednisolone 21-phosphate disodiumsalt, methylprednisolone 21-succinaate sodium salt, paramethasonedisodium phosphate and prednisolone 21-succinate sodium salt, andanticoagulants, such as citric acid, citrate salts (e.g., sodiumcitrate), dextrin sulfate sodium, aspirin and EDTA.

In yet another embodiment of the invention, the coating formulationincludes a solubilizing/complexing agent, which can compriseAlpha-Cyclodextrin, Beta-Cyclodextrin, Gamma-Cyclodextrin,glucosyl-alpha-Cyclodextrin, maltosyl-alpha-Cyclodextrin,glucosyl-beta-Cyclodextrin, maltosyl-beta-Cyclodextrin, hydroxypropylbeta-cyclodextrin, 2-hydroxypropyl-beta-Cyclodextrin,2-hydroxypropyl-gamma-Cyclodextrin, hydroxyethyl-beta-Cyclodextrin,methyl-beta-Cyclodextrin, sulfobutylether-alpha-cyclodextrin,sulfobutylether-beta-cyclodextrin, andsulfobutylether-gamma-cyclodextrin. Most preferredsolubilizing/complexing agents are beta-cyclodextrin, hydroxypropylbeta-cyclodextrin, 2-hydroxypropyl-beta-Cyclodextrin andsulfobutylether7 beta-cyclodextrin.

In another embodiment of the invention, the coating formulation includesat least one non-aqueous solvent, such as ethanol, isopropanol,methanol, propanol, butanol, propylene glycol, dimethysulfoxide,glycerin, N,N-dimethylformamide and polyethylene glycol 400.

Preferably, the coating formulations have a viscosity less thanapproximately 500 centipoise and greater than approximately 3centipoise.

In one embodiment of the invention, the thickness of the biocompatiblecoating is less than 25 microns, more preferably, less than 10 microns,as measured from the microprojection surface.

The following examples are given to enable those skilled in the art tomore clearly understand and practice the present invention. They shouldnot be considered as limiting the scope of the invention but merely asbeing illustrated as representative thereof. A method has been devisedto calculate the distribution of ionic species in polypeptides and otherelectrolytes. Equations for equilibrium calculations have been availablefor many years. They are based on the classic equilibrium laws. They canbe used successfully to calculate the net charge of polyelectrolytessuch as polypeptides as well as the pI of a protein. Net charge and pIcalculations are powerful tools for characterizing and purifyingpolypeptides. Nevertheless, these calculations do not yield directinformation about the species present in solution at a specific pH. Forexample, the pH range in which species with suspected low solubility arepresent are not predicted from these methods. Various attempts have beenmade to estimate the equilibria between different ionic forms inpolyelectrolytes. These attempts have been summarized by Edsall J. T.(Proteins as acids and bases, in proteins, amino acids and peptides asions and dipolar ions, Cohn E. J. & Edsall J. T. eds; Hafner Pub; NewYork and London, 1943, 444-505).

The most successful approach describes a probability distributionfunction for a system of independently ionizing groups. In thistreatment, the various groups are classified by classes, eachcorresponding to one pKa value. The procedure is somewhat cumbersome andis not easily amenable to automatic computation. In addition,calculations are limited to the net charged species and do not includedescription of the charge distribution within the molecule.Surprisingly, with polyelectrolytes, very little attention has been paidto the concentrations of the actual species that are present insolution. This seems to be the result of the lack of equationsdescribing the distribution of species in the presence of overlappingpK_(a) values, that is, two or more pK_(a) values separated by less thanabout 3 pH units. In this case, approximations are being used tocalculate the distribution of the species. In a polypeptide molecule,where more than ten overlapping pK_(a) values is commonplace,computations based on these approximations are not practical and wouldcertainly yield erroneous results. As a result, distribution of speciesin polypeptides apparently has not been described. A method has beendevised that provides equations describing the species distribution forany polyelectrolyte, provided that their pK_(a) values are known. Acomputational algorithm for performing these calculations is alsoprovided.

Methods

For polypeptides, the acid-base radicals implicated and their pK_(a)values are, respectively: terminal carboxyl, pK_(a)=3.05; β-carboxyl ofaspartate, pK_(a)=3.93; γ-carboxyl of glutamate, pK_(a)=4.43; thiol ofcysteine, pK_(a)=8.38; phenol of tyrosine, pK_(a)=10.36; imidazolium ofhistidine, pK_(a)=5.96; terminal ammonium, pK_(a)=8.1; ε-ammonium oflysine, pK_(a)=10.59; guanidinium of arginine, pK_(a)=12.48. The abovepK_(a) values are averages compiled from the literature and used in theexamples. The pI values were extrapolated from the net charge profile ofthe molecule calculated from their pK_(a) values.

Determination of the Species Concentrations in a Polyelectrolyte:

For a weak acid, AH, the equilibrium can be writtenAH

A⁻+H⁺

Its dissociation constant being:K _(a)=(A ⁻)×(H ⁺)/(AH)

(A⁻), (H⁺), and (AH) being the respective concentrations of the species.

From the above, the classic Henderson-Hasselbalch equation can bederived:pH=pK _(a)+Log((A ⁻)/(AH))

Assuming that: (A⁻)+(AH)=1, this equation yields:Mole fraction neutral=1/(1+10^(pH-pKa))=Pwhich can also be defined as the probability of the acid to be neutral.Similarly:Mole fraction ionized, negatively charged=1−1/(1+10^(pH-pKa))=1−PNet charge=1/(1+10^(pH-pKa))−1

For a weak base, B, the equilibrium can be written:B+H⁺

BH⁺

Its dissociation constant being:K _(a)=(B)×(H ⁺)/(BH ⁺)

Similarly:pH=pK _(a)−Log(BH ⁺ /B),Mole fraction neutral=1/(1+10^(pKa-pH))=QMole fraction ionized, positively charged=1−1/(1+10^(pKa-pH))=1−QNet charge=1−1/(1+10^(pKa-pH))

The species are defined as all the possible combinations of the chargesfor the acidic functions and basic functions of the compound insolution. For example if the compound presents only acidic functions,the species take the values like 0⁻, 1⁻, 2⁻, and etc. Similarly, if thecompound presents only basic functions, the species take the values like0⁺, 1⁺, 2⁺, and etc. If the compound has both acidic and basicfunctions, then the species take the values of 0⁻0⁺, 0⁻1⁺, 1⁻0⁺, 1⁻1⁺,etc. The net charged species are defined as the sum of all speciespresenting an identical net charge. They take the values: . . . −2, −1,0, +1, +2 . . . .

For a compound bearing one acidic (negatively charged) pK_(a), thespecies present in solution at any pH are: 0⁻ and 1⁻ (one species isneutral: no positive charge and no negative charge; the other specieshas one negative charge and no negative charge). P₁ being theprobability of the acidic group to be neutral, the mole fraction ofthese species at a specific pH is:

0⁻: P₁

1⁻: 1-P₁

For a compound bearing one acidic pK_(a), and one basic (positivelycharged) pK_(a), the species present in solution at a specific pH are:0⁻0⁺, 0⁻1⁺, 1⁻0⁺, 1⁻1⁺¹.

P₁ and Q₁ being the probability of the acidic and basic group,respectively, to be neutral, the mole fraction of these species at aspecific pH is:

0⁻0⁺: P₁×Q₁

0⁻1⁺: P₁×(1−Q₁)

1⁻0⁺: (1−P₁)×Q₁

1⁻1⁺: (1−P₁)×(1−P₁)

For a compound bearing one acidic pK_(a), and two basic pK_(a), thespecies present in solution at any pH are: 0⁻0⁺, 0⁻1⁺, 0⁻2⁺, 1⁻0⁺, 1⁻1⁺,1⁻2⁺.

P₁ being the probability of the acidic groups to be neutral, and Q₁ andQ₂ being the probabilities of the basic groups to be neutral, the molefraction of these species at a specific pH is:

0⁻0⁺: P₁×Q₁×Q₂

0⁻1⁺: (P₁×Q₁×(1−Q₂))+(P₁×(1−Q₁)×Q₂)

0⁻2⁺: P₁×(1−Q₁)×(1−Q₂)

1⁻0⁺: (1−P₁)×Q₁×Q₂

1⁻1⁺: ((1−P₁)×Q₁×(1−Q₂))+((1−P₁)×(1−Q₁)×Q₂)

1⁻2⁺: (1−P₁)××(1−Q₁)×(1−Q₂)

Etc. . . .

As can be seen, there are (N+1) (M+1) species, N and M being the numberof acidic and basic pK_(a), respectively. In the previous example, therewere six possible species. The possible net charged species, which are−1, 0, +1, +2. The number of possible net charged species is (N+M+1).The mole fraction of these net charged species at a specific pH can beeasily deduced. Using the preceding example:

−1: (1−P₁)×Q₁×Q₂

0: (P₁×Q₁×Q₂)+((1−P₁)×Q₁×(1−Q₂))+((1−P₁)×(1−Q₁)×Q₂)

+1: (P₁×Q₁×(1−Q₂))+(P₁×(1−Q₁)×Q₂)+(1−P₁)××(1−Q₁)×(1−Q₂)

+2: P₁×(1−Q₁)×(1−Q₂)

Computational Algorithm of the Species and Valences of aPolyelectrolyte:

Based on the above equations, an algorithm has been derived which isused to calculate the charge, net charge, species and valences presentin a polyelectrolyte. In the following, a bold and upper letter todenote a vector or a matrix, and a lower letter represent an element ofthe vector or the matrix.

Suppose we know that there are N acidic functions and M basic functionsin the compound, their pK_(a) values are given, and the pH value of thesolution is also given. Let PKA_(a) be the N by 1 vector of acidicpK_(a) values, and PKA_(b) be the M by 1 vector of basic pK_(a) values:

PKA_(a)=(pKa_(a1), pKa_(a2), . . . , pKa_(aN))^(T)

PKA_(b)=(pKa_(b1), pKa_(b2), . . . , pKa_(bM))^(T)

P=(p₁, p₂, . . . , p_(N))^(T)

Q=(q₁, q₂, . . . , q_(M))^(T)p _(i)=1/(1+10^(pH-pKa) ^(ai) )  (1)q _(j)=1/(1+10^(pKa) ^(bj) ^(−pH))  (2)

where P and Q are mole fraction neutral for acidic components and basicfunctions, respectively. They also can be understood as theprobabilities of being neutral for either acid or base. Let CHARGE_(a)denote the N by 1 vector of charge for the acids, while CHARGE_(b) isthe M by 1 vector for the bases:

CHARGE_(a)=(charge_(a1), charge_(a2), . . . , charge_(aN))^(T)

CHARGE_(b)=(charge_(b1), charge_(b2), . . . , charge_(bM))^(T)charge_(ai)=1/(1+10^(pH-pKa) ^(ai) )−1  (3)charge_(bj)=1−1/(1+10^(pKa) ^(bj) ^(−pH))  (4)

$\begin{matrix}{{{net}\mspace{14mu}{charge}} = {{\sum\limits_{i = 1}^{N}{charge}_{ai}} + {\sum\limits_{j = 1}^{M}{charge}_{bj}}}} & (5)\end{matrix}$where net charge is the charge of the complex molecule in the solution.

Next, let us consider the species of the molecule compound. Forsimplicity, we will use α to represent the species. In order tounderstand the species calculation algorithm, let's start from thesimple case. Suppose the compound only has N acids, we want theprobabilities of α in the solution. Based on the above derivation, P isthe probability vector for the acids being neutral. Let us consider astatistical experiment. Suppose that the compound in the solution ismade by adding one acid by one acid. At the beginning, when only oneacid is in the solution, we have:Prob(α=0⁻,1 acid)=p ₁  (6)Prob(α=1⁻,1 acid)=1−p ₁  (7)Prob(α=2⁻,1 acid)= . . . ==Prob(α=N ⁻,1 acid)=0  (8)

Then given that we already have i acids in the solution, and add onemore thereafter. The relationships of the probabilities are:Prob(α=0⁻ ,i+1 acids)=Prob(α=0⁻ ,i acids|the (i+1)th acid=0)Prob(the i+1th acid=0)Prob(α=j ⁻ ,i+1 acids)=Prob(α=j ⁻ ,i acids|the (i+1)th acid=0)Prob(the i+1th acid=0)+Prob(α=(j−1)−,i acids|the (i+1)th acid=1)Prob(the i+1th acid=1)

Here we are making an assumption that all the acids are independent,hence we can rewrite the above equations:Prob(α=0⁻ ,i+1 acids)=Prob(α=0⁻ ,i acids)Prob(the i+1th acid=0)  (9)Prob(α=j ⁻ ,i+1 acids)=Prob(α=j ⁻ ,i acids)Prob(the i+1th acid=0)+Prob(α=(j−1)−,i acids)Prob(the i+1th acid=1)  (10)

Equation (9) and (10) give us an easy way to calculate theprobabilities. To implement them, let R be a N+1 by N matrix:r[j,i]=Prob(α=(j−1)⁻ ,i acids)

We can rewrite (6), (7), (8), (9) and (10) as:r[1,1]=P₁  (11)r[2,1]=1−P ₁  (12)r[3,1]= . . . =r[N+1,1]=0  (13)r[1,i+1]=r[1,i]p _(i+1)  (14)r[j+1,i+1]=r[j+1,i]p _(i+1) +r[j,i](1−p _(i+1))  (15)i=1 . . . (N−1), j=1, . . . , N

It is very straightforward to code the above recursion algorithm byloops, and the last column of R simply represents the probabilities ofspecies when a compound with N acids is in the solution. Without losingof the generality, let A be the last column of R. Similarly let B be thespecies probability vector when a compound of M bases is in thesolution, and the dimension is M+1 by 1. The calculation of B followsthe same rule as A. If the compound has N acids and M bases, theprobabilities of species are:C=A×B ^(T)  (16)c[i,j]=Prob(α=(i−1)⁻(j−1)⁺)  (17)i=1,2, . . . , N+1j=1,2, . . . , M+1where C is an N+1 by M+1 matrix. At last, the net charged species (β)can be constructed based on C:

$\begin{matrix}{{{{Prob}\mspace{11mu}\left( {\beta = i} \right)} = {\sum\limits_{\underset{\underset{{j = 1},\ldots,{N + 1}}{{k = 1},\ldots,{M + 1}}}{i = {k - j}}}{c\left\lbrack {j,k} \right\rbrack}}}{{{{where}\mspace{14mu} i} = {- N}},\ldots\mspace{11mu},{- 1},0,1,\ldots\mspace{11mu},M}} & (18)\end{matrix}$

Based on the above, the distribution of charged or neutral species forselected compounds can be calculated, which is illustrated in thefollowing examples.

Example 1

FIG. 1 shows the charge profile of acetic acid (pKa 4.75) as a functionof pH. At pH below about 2.5 the carboxyl group of the acetic acid iscompletely protonated and thus there is no charge on the molecule. Asthe pH increases from about 2.5 to about 7, more and more of thecarboxyl moieties become ionized and thus forming the negatively chargedacetate ion. At about pH 7, all of the carboxyl groups are ionized.

FIG. 2 shows the mole ratios of acetic acid and acetate. At pH 0, withthe carboxyl group of acetic acid fully protonated, there essentiallyonly acetic acid, thus the mole fraction is 1. At about ph 2.5, thereionization of the carboxyl group begins and the solid curve representingacetic acid in graph starts to move downward. At the same time, thedashed line, representing the ionized acetate, starts to move upwardsoff of the 0.00 line. At about pH 4.7 there are equal numbers of chargedand uncharged moieties. At pH greater than about 7, there is no longerany uncharged acetic acid and essentially all of species are the chargedacetate ion.

Many drugs exhibit maximum solubility in water when they are in anelectrically charged state. FIG. 3 shows the charge profile of fentanyl,a small molecular weight weakly basic drug presenting one basic pKa,8.5. At pH below 6, essentially all of the fentanyl is positivelycharged, while at pH above 11, essentially all of the fentanyl isneutral.

FIG. 4 shows the mole ratios of the neutral (Fentanyl base-solid line)and charged fentanyl (Fentanyl⁺¹-dashed line) species at different pHs.From pH 0 to about pH 6, there is essentially no Fentanyl base presentand 100% is the charged Fentanyl⁺¹. From pH about 6 to about pH 11,there is a transition. The Fentanyl⁺¹ decreases at the same rate thatthe Fentanyl base increases. At or above pH 11, essentially all of theFentanyl exists in the non-charged, neutral, Fentanyl base.

Complex molecules such as peptides and proteins also exhibit chargecharacteristics that are dependant on pH. FIG. 5 shows the chargeprofile of hPTH(1-34), a peptide presenting 11 basic pKa's, and sixacidic pKa's. At pH 9, the peptide presents a zero net electric charge.This point is also called the isoelectric point or pI.

FIG. 6 shows the mole ratios of the net charged species of PTH. Thespecies range from a +11 charge to a −6 charge. The neutral species onlyexist in significant amounts in the pH range of about 6 to about 11.5.In this pH range, PTH precipitates out of solution.

FIG. 7 shows the mole ratios for fentanyl acetate (dashed line), aceticacid (solid line), and the neutral form of fentanyl (fentanylbase-dotted line). These are the species that are present in solution atdifferent pH's when various ratios of fentanyl base and acetic acid aremixed in solution. The pH of fentanyl acetate (mole ratio 1 to 1) insolution is predicted to be about 6.6. At that pH, about 1% of fentanylis present as fentanyl base, which, for a 10 mg/mL solution totalfentanyl, would be at or above the limit of solubility of the base,which would therefore precipitate out. Solubilization can be achieved bysupplementing the formulation with excess acetic acid, which will resultin acidification of the formulation and will therefore results in adecrease in the amount of fentanyl base. Nevertheless, during drying andsubsequent storage the free acetic acid will evaporate which willineluctably result in the formation of the water insoluble base.Subsequent reconstitution in water would not allow total solubilizationof fentanyl. The use of a non-volatile counterion would provide a solidsoluble formulation of fentanyl as long as the pH is maintained at least2 pH units, preferably 3 pH units, below the pKa of fentanyl. This couldbe achieved by providing at least a slight excess of non-volatilecounterion to the formulation (ie. a mole ration of non-volatilecounterion to fentanyl slightly greater than 1:1). In addition, volatilecounterions could be added to that formulation without affecting thesolubility of the dry coating.

FIG. 8 shows the mole ratios for acetic acid (solid line) and theneutral form of hPTH(1-34) (dotted line). The pH of a hPTH(1-34)hexaacetate (mole ratio 1 to 6) in solution is predicted to be about 5(see FIG. 5). At that pH, negligible amounts of hPTH(1-34) are presentas hPTH(1-34) zero net charge (PTH 0—see the charge curve for the “0”charge species in FIG. 6) and hPTH(1-34) is highly soluble in water atconcentrations in excess of 20%. As in the case of fentanyl, duringdrying and subsequent storage, the volatile free acetic acid willevaporate which will result in a shift to a higher pH, which results information of the water insoluble PTH 0. Subsequent reconstitution inwater would not allow total solubilization of hPTH(1-34). The use of anon-volatile counterion would provide a solid soluble formulation ofhPTH(1-34) as long as the pH is maintained at least 2.5 pH units,preferably 3 pH units, below the pI of hPTH(1-34). This could beachieved by providing at least about 2 non-volatile counterions to eachmolecule of hPTH(1-34). As in the case of fentanyl, volatile counterionscould be added to that formulation without affecting the solubility ofthe dry coating.

Example 2

Several aqueous formulations containing hPTH(1-34) were prepared. Theseformulations contained the volatile counterion acetic acid. Severalformulations contained additional non-volatile counterions hydrochloricacid, glycolic acid, or tartaric acid (see Table 1). Microprojectionarrays (microprojection length 200 μm, 595 microprojections per array)had a skin contact area 2 cm². The tips of the microprojections werecoated with these formulations by passing the arrays over a rotatingdrum carrying the hPTH(1-34) formulations using the method and apparatusdisclosed in U.S. patent application Ser. No. 10/099,604 filed Mar. 15,2002, which is hereby incorporated by reference in its entirety. Foursuccessive coatings were performed on each microprojection array at 2-8°C. The amount of peptide coated on the arrays was evaluated byultraviolet spectroscopy at a wavelength of 275 nm. Scanning electronmicroscopy revealed that the solid coating had a very smooth surfacewith no evidence of cracking. Furthermore good uniformity of coatingfrom microprojection to microprojection was observed, with the coatinglimited to the first 100 μm of the microprojection tip. Some of thetip-coated arrays were subsequently used for drug delivery studies inhairless guinea pigs (HGPs).

The HGPs were anesthetized by intramuscular injection of xylazine (8mg/kg) and ketamine HCl (44 mg/kg). The anesthetized HGPs werecatheterized through the carotid artery. The catheter was flushed withheparinized saline (20 IU/mL) to prevent clotting. Animals weremaintained under anesthesia throughout the experiment via injection ofsodium pentobarbital (32 mg/mL) directly into the catheter (0.1mL/injection). Before application of the coated microprojection arrays,blood samples were taken into heparinized vials (final concentration ofheparin at 15 IU/mL), which served as 0 or baseline samples.

The application of the coated microprojection arrays was performed onthe flank of the anesthetized animals with a spring-driven impactapplicator (total energy=0.4 Joules, delivered in less than 10milliseconds), the type disclosed in U.S. patent application Ser. No.09/976,798 filed Oct. 12, 2001, which is hereby incorporated byreference in its entirety. The system applied comprised a coatedmicroprojection array device, adhered to the center of a LDPE backingwith an adhesive (7 cm2 disc). Patches were remained on the skin for 1 h(n=4-5). A group of animals (n=5) received an intravenous injection of22 μg hPTH(1-34) instead of the microprojection array. Blood sampleswere collected through the carotid catheter at time intervals followingpatch application or IV injection. All blood samples were centrifugedimmediately for plasma collection, which was then stored at −80° C.until analysis. Plasma hPTH(1-34) was determined by the EIA, acommercial enzyme immunoassay kit for hPTH(1-34) from Peninsula Lab.(San Carlos, Calif.). The hPTH(1-34) dose delivered by microprojectionarrays was extrapolated based on the area under the curve (AUC)calculation compared to IV administration of hPTH(1-34).

Results are shown in Table 1, which demonstrate that different amountsof HPTH(1-34) were delivered from each solid formulation. The solidformulations containing only hPTH(1-34) acetate (No. 1 and 2) deliveredless than 2 μg on average. Addition of non-volatile counterions tohPTH(1-34) acetate increased delivery significantly to up to 11.2 μgafter the addition of the non-volatile counterion glycolic acid (No. 5).The two other non-volatile counterions tested, tartaric (No. 6) andhydrochloric acid (Nos. 3 and 4) also increased hPTH(1-34) delivery.

TABLE 1 PTH formulations and hPTH(1-34) delivery in the hairless guineapig Ratio Amount of (hPTH(1-34):Acetate:non- hPTH(1-34) Amount volatilecoated on array delivered No Formulation solution (wt %) counterion)(μg) ± SD (μg) ± SD 1 21.2% hPTH(1-34), 1:3:0 28.0 ± 6.6  1.1 ± 1.1 3.8%acetic acid, water q.s. 2 21.2% hPTH(1-34), 1:3:0 35.0 ± 11.4 1.5 ± 1.73.8% acetic acid, water q.s 3 22.3% hPTH(1-34), 1:2:2 40.0 ± 9.8  5.9 ±2.5 2.7% acetic acid, 0.4% HCl, water q.s. 4 16.2% hPTH(1-34), 1:3:330.5 ± 2.3  6.1 ± 4.0 3.8% acetic acid, 0.5% HCl, 20.2% excipients,water q.s. 5 6.2% hPTH(1-34), 1:3:4 45.9 ± 11.7 11.2 ± 2.7  3.8% aceticacid, 2.1% glycolic acid, 12.2% excipients, water q.s. 6 16.2%hPTH(1-34), 1:3:2 29.0 ± 4.3  4.2 ± 1.5 3.8% acetic acid, 1.2% Tartaricacid, 20.23% excipients, water q.s.

Example 3

In order to demonstrate depletion of coatings containing volatilecounterions, we coated 1 cm² titanium disc with a pH 5 aqueousformulation made by solubilizing 20 wt % of the acetate form of apeptide, an hGRF analog. Following coating, each disc was stored under20 mL nitrogen atmosphere at room temperature for 3 months.

Results in Table 2 compare the acetate/peptide mole ratio at the initialand the 3 month time points. At the initial time point for one mole ofpeptide there are 6.5 moles of acetic acid. At pH 5, the peptidepresents about 4.5 positive charges (see FIG. 9), leaving 2 moles offree acetic acid per mole of peptide. After storage for 3 months atambient conditions the number of moles of acetate per mole of peptidedecreases to 3.8, demonstrating depletion of the volatile counterionfrom the coating. Extrapolation from the charge profile of FIG. 9 showsthat reconstitution of the coating in water would yield a pH 9.5solution, which represents a dramatic increase in pH from the pH of theinitial solution.

TABLE 2 Acetate/hGRF analog mole ratio in Ti coated samples DrugAcetate/peptide Storage content Average coating mole ratio Samplecondition (μg/cm2) thickness (μm) (Initial = 6.5) Ti disc 3 monthsRoom384 3.8 3.8 ± 0.1 temperature N² atmosphereWithout departing from the spirit and scope of this invention, one ofordinary skill can make various changes and modifications to theinvention to adapt it to various usages and conditions. As such, thesechanges and modifications are properly, equitably, and intended to be,within the full range of equivalence of the following claims.

1. A composition comprising a formulation of a pharmaceutically active agent, a volatile counterion and a non-volatile counterion, wherein said pharmaceutically active agent is hPTH(1-34) present as uncharged species and as charged species and wherein said non-volatile counterion is present in an amount sufficient to achieve a molar ratio between said uncharged species and charged species of about 1 uncharged species to at least 100 charged species; wherein said formulation has increased pH stability and solubility when dried, and wherein said composition is applied to a transdermal delivery device having stratum corneum-piercing microprojections; wherein said composition achieves an increase in delivery of the pharmaceutically active agent compared to a composition comprising a formulation without said non-volatile counterion.
 2. The composition of claim 1, wherein said formulation has a pH, said pharmaceutically active agent has a positive charge at said formulation pH and said non-volatile counterion comprises a strong acid.
 3. The composition of claim 2, wherein said strong acid has at least one pKa lower than about
 2. 4. The composition of claim 3, wherein said strong acid is selected from the group consisting of hydrochloric acid, hydrobromic acid, nitric acid, sulfonic acid, sulfuric acid, maleic acid, phosphoric acid, benzene sulfonic acid and methane sulfonic acid.
 5. The composition of claim 1, wherein said pharmaceutically active agent has a mole ratio between uncharged species and charged species of at least 1 to 100 at a pH between 4 and
 10. 6. The composition of claim 1, wherein said composition achieves a five fold increase in delivery of the pharmaceutically active agent compared to a composition comprising a formulation without said non-volatile counterion.
 7. A composition comprising a formulation of a pharmaceutically active agent, a volatile counterion and a non-volatile counterion, wherein said pharmaceutically active agent is hPTH (1-34) present as uncharged species and as charged species; wherein said pharmaceutically active agent has a positive charge at the pH of the formulation and said non-volatile counterion comprises a strong acid; and wherein said formulation has increased pH stability and solubility when dried.
 8. The composition of claim 1, wherein the non-volatile counterion comprises a weak acid or a strong acid.
 9. The composition of claim 8, wherein the weak acid is glycolic acid or tartaric acid.
 10. The composition of claim 8, wherein the strong acid is hydrochloric acid.
 11. The composition of claim 8, wherein hPTH(1-34) is present as an acetate salt.
 12. A composition comprising a formulation of hPTH(1-34), a volatile counterion and a non-volatile counterion from a weak or strong acid, wherein said non-volatile counterion is present in an amount sufficient to achieve a molar ratio of about 1 uncharged hPTH species to at least about 100 charged hPTH species, wherein said composition achieves an increase in delivery of said hPTH when applied to a transdermal delivery device having stratum corneum-piercing microprojections compared to a composition comprising a formulation without said non-volatile counterion.
 13. The composition of claim 12, wherein the weak acid is glycolic acid or tartaric acid.
 14. The composition of claim 12, wherein the strong acid is hydrochloric acid.
 15. The composition of claim 12, wherein hPTH(1-34) is present as an acetate salt. 